Worm Breeder's Gazette 11(1): 69
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
At the meeting in May there was considerable interest in the protocol we have been using to obtain nuclear extracts. The procedure we use to obtain the nuclei is that described by Dixon et al (WBG 2 (3) :73-74) except that PMSF is present at 1 mM in all buffers and 1 mM DTT is used in place of mercaptoethanol. The efficiency depends in great part on the tightness of the homogenizer used to shear the worms. The nuclei are washed twice in 10 ml of TKM (50 mM Tris-HCI (7.5), 25 mM KCL and 5 mM MgCl2) and resuspended in 3 volumes of TKM. At this point the volume of nuclei is sufficiently small (0.05-0.2 ml) to carry out the remaining steps in microfuge tubes. EDTA is added to 0. 01 N and an equal volume of 2X Iysis buffer (1X= 40 mM Tris-HCI (7.5), 1 mM EDTA, 0.5 mM DTT, 1 M NaCl, 10% glycerol) is added. Tubes are mixed gently for 5 minutes in the cold and one-half volume of 18% PEG8000 in 1X Iysis buffer is added slowly with gentle mixing. The tubes are slowly rotated in the cold for 1 hour and nucleic acids are removed by centrifugation (17000 G for 20 minutes). This crude supernatant appears to be free of nucleases and proteases and can be used directly or dialyzed against your favorite buffer using micro dialysis with membranes having a 12 kD cut-off, protein concentration decreases by a factor of about 2 during dialysis. Two grams of worms yields 0.5 to 1 mg of protein before dialysis. The extracts have little contamination by cytoplasmic components as the vitellogenins appear to be absent when SDS-PAGE is performed. Using extracts from mixed stage and sex N2s, we have repeated the gel retardation experiments which we described at the meeting. Specific binding can be demonstrated with a double-stranded substrate containing the TATTGAAA sequence which we have suggested is an X- linked binding site used in X/A ratio assessment. This binding is competed by double-stranded oligos which contain the octamer but not by those which lack it. The negatives include a sequence which precisely deletes the 8 base pairs but leaves 56 nucleotides around the site intact. Single stranded oligonucleotides, including the two complementary strands annealed to give a strong specific competitor do not compete for binding. Curiously, the 8 bp sequence by itself does not compete even when ligated into a large concatemer despite the fact that it is feminizing in the microinjection assay which we have previously described (WBG 10 (1): 62-63).